The present invention relates to novel fungal host cells and to methods of producing proteins. More specifically, the invention relates to a host cell useful for the expression of heterologous proteins in which the host cell has been genetically modified in order to express significantly reduced levels of a metalloprotease and an alkaline protease. Moreover, the invention relates to a method of producing proteins of interest in high yields by using the fungi of the invention, in which the method comprises cultivating the host cell in a suitable growth medium, followed by recovery of the desired protein. The invention also comprises methods for producing such fungi and DNA constructs to be used in these methods.
The use of recombinant host cells in the expression of heterologous proteins has in recent years greatly simplified the production of large quantities of commercially valuable proteins which otherwise are obtainable only by purification from their native sources. Currently, there is a varied selection of expression systems from which to choose for the production of any given protein, including eubacterial and eukaryotic hosts. The selection of an appropriate expression system often depends not only on the ability of the host cell to produce adequate yields of the protein in an active state, but, to a large extent, may also be governed by the intended end use of the protein.
One problem frequently encountered is the high level of proteolytic enzymes produced by a given host cell or present in the culture medium. It has been suggested that one could provide host organisms deprived of the ability to produce specific proteolytic compounds. For example, International Patent Application WO 90/00192 (Genencor) describes filamentous fungal hosts incapable of secreting enzymatically active aspartic proteinase, and EP 574 347 (Ciba Geigy AG) describes Aspergillus hosts defective in a serine protease of the subtilisin-type.
Metalloproteases have been isolated from a number of eukaryotic sources. Neutral metalloproteases, i.e. metalloproteases having optimal activity at neutral pH, isolated from strains of Aspergillus also have been reported. The physicochemical properties of two neutral metalloproteases from Aspergillus sojae, NpI and NpII, have been reported in a series of studies (Sekine, H. 1972. Agric. Biol. Chem. 36:198-206, 207-216; Sekine, H. 1972. Agric. Biol. Chem. 36:2143-2150). It was revealed that the enzymatic and physicochemical characteristics of NpI, but not Np II, resemble those of Bacillus thermoproteolyticus thermolysin. Recently, the cDNA sequence of a neutral metalloprotease, NpII, from Aspergillus oryzae was disclosed (Tatsumi H, et al. 1991. Mol. Gen. Genet. 228:97-103). However, the cDNA sequence of a neutral metalloprotease from the NpI group from Aspergillus oryzae has never been disclosed.
Alkaline protease is a serine protease with an alkaline pH optimum (Nakagawa, Y. 1970. Methods Enzymol. 19:581-591). It is a homologue of subtilisin and is the predominant extracellular alkaline endopeptidase of Aspergillus oryzae. The gene has been isolated and characterised (Murakami, et al. 1991. Agric. Biol. Chem. 55:2807-2811). Two other species of Aspergillus, A. flavus and A. sojae, produce identical or closely related enzymes.
A potential role of metalloproteases and alkaline proteases in reducing the stability of protein products obtained from Aspergillus has not been reported.
According to the present invention it has now been found that the proteolytic activity of neutral metalloprotease I and alkaline protease, either individually or in combination, may significantly reduce the stability of a protein product of interest produced by a cell resulting in a reduced yield of the said protein.
Accordingly, the present invention provides a host cell useful for the expression of a heterologous protein product, in which the cell has been genetically modified in order to express significantly reduced levels of both a metalloprotease and an alkaline protease, by comparison to the parental cell.
In another aspect, the invention provides a method of producing a heterologous protein product in a host cell of the invention, in which the method comprises introducing into the host cell a nucleic acid sequence encoding the protein, cultivating the host cell in a suitable growth medium, and recovering the heterologous protein product.
By the method of the invention, proteolytic activity resulting from a neutral metalloprotease I and an alkaline protease are significantly reduced, thereby improving the stability and yield of the protein obtained by the method. Moreover, the protein obtained by the method of the invention may be a precursor protein such as a zymogen, a hybrid protein, a protein obtained as a pro sequence or pre-pro sequence, or any other type of immature form.